describe and carry out investigations using whole plants, including aquatic plants, to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis

Photosynthesis – Investigation of Limiting Factors (Cambridge International AS & A Level Biology 9700 – Topic 13.2)

Learning Outcomes (as stated in the syllabus)

• Describe and carry out investigations using whole plants, including aquatic plants, to determine the effects of light intensity, carbon‑dioxide concentration and temperature on the rate of photosynthesis.
• Analyse experimental data to identify which factor is limiting under the conditions used.
• Evaluate the reliability of the investigation and suggest improvements.

These outcomes address Assessment Objective 2 (knowledge & data handling) and Assessment Objective 3 (experimental skills and evaluation).

1. Syllabus Coverage – What Must Be Included

2. Theoretical Background

2.1 Light‑dependent Reactions

• Location: Thylakoid membranes of the chloroplast.
• Key components: Photosystem II (PS II), plastoquinone (PQ), cytochrome b₆f complex, plastocyanin (PC), Photosystem I (PS I), ferredoxin (Fd), NADP⁺ reductase.
• Z‑scheme: Electrons flow from H₂O (via the oxygen‑evolving complex) → PS II → PQ → cytochrome b₆f → PC → PS I → Fd → NADP⁺, producing NADPH and a proton gradient that drives ATP synthesis (photophosphorylation).
• Cyclic photophosphorylation: Electrons from Fd return to the PQ pool, generating extra ATP without NADPH formation – important when the Calvin cycle demands more ATP than NADPH.
• Products per 2 photons (non‑cyclic): 2 H₂O → O₂ + 4 e⁻ + 4 H⁺; 2 NADP⁺ + 2 H⁺ → 2 NADPH; ≈3 ATP (via chemiosmosis).

2.2 Light‑independent Reactions (Calvin Cycle)

• Location: Stroma of the chloroplast.
• Enzyme: Ribulose‑1,5‑bisphosphate carboxylase/oxygenase (Rubisco).
• Three phases:

  1. Carbon fixation: CO₂ + RuBP → 2 3‑PGA.
  2. Reduction: 3‑PGA + ATP + NADPH → G3P (glyceraldehyde‑3‑phosphate).
  3. Regeneration: 5 G3P + ATP → 3 RuBP (ready for the next turn).

• Overall stoichiometry (per 6 CO₂): 6 CO₂ + 6 H₂O + 18 ATP + 12 NADPH → C₆H₁₂O₆ + 6 O₂ + 18 ADP + 12 NADP⁺ + 18 Pᵢ.
• Limiting factors: Availability of ATP/NADPH (light‑dependent), Rubisco activity (temperature & CO₂), and substrate CO₂ concentration.

3. Investigation Overview

The three single‑factor investigations below each vary one environmental variable while keeping the other two constant. Whole aquatic plants (e.g., Elodea canadensis) are preferred because O₂ evolution can be collected directly in an inverted graduated cylinder.

4. Variables in the Experiments

5. General Experimental Design

1. Select a healthy whole plant or a fresh leaf. For aquatic plants, place the sprig in a transparent, airtight container filled with distilled water.
2. Introduce the independent variable:

   • Light intensity: Adjustable lamp, neutral‑density filters, or set distances; verify with a lux meter or quantum sensor.
   • CO₂ concentration: Inject known volumes of CO₂ using a syringe or gas burette; mix gently.
   • Temperature: Thermostatically controlled water bath (±0.5 °C).

3. Collect the evolved O₂ in an inverted graduated cylinder (or gas syringe) and record the volume at regular intervals (e.g., every minute for 10 min).
4. Repeat each treatment at least three times; randomise the order of trials to minimise systematic error.
5. Calculate the rate (slope of the linear portion of the volume‑time graph) and analyse as described in the specific sections below.

6. Investigating Light Intensity

6.1 Apparatus

• Elodea sprigs (≈2 cm long) or a fresh spinach leaf.
• Transparent water bath with an inverted 100 mL graduated cylinder.
• Adjustable lamp (60 W fluorescent) with a dimmer or a set of calibrated distances.
• Lux meter (or quantum sensor) – optional but recommended.
• Thermometer (±0.5 °C) to monitor water temperature.
• Stopwatch.

6.2 Method (single‑factor design)

1. Fill the water bath with distilled water at 25 °C and insert the plant.
2. Set the lamp at a chosen distance, record the light intensity (e.g., 500 lux).
3. Start the timer; record O₂ volume every minute for 10 min.
4. Change the lamp distance or dimmer setting to obtain a higher intensity (e.g., 1000, 1500, 2000 lux) and repeat steps 2–4.
5. Maintain water temperature within ±1 °C for all trials.
6. Perform three replicates per intensity; randomise the order of intensities.

6.3 Sample Data Table

6.4 Analysis

• Plot O₂ volume (y) against time (x) for each intensity; the slope of the linear region = rate (mL min⁻¹).
• Plot the calculated rates against light intensity.

  • Initial linear increase → light‑limited region.
  • Plateau → light‑saturation point (maximal capacity of the light‑dependent reactions).
  • Decline at very high intensities → photoinhibition (damage to PS II).

• Explain the curve using photon absorption, ATP/NADPH production, and the capacity of the Calvin cycle.

7. Investigating Carbon‑Dioxide Concentration

7.1 Apparatus

• Elodea sprigs in a sealed gas‑tight chamber.
• Three‑way stopcock for gas inlet/outlet.
• Syringe or gas burette to deliver known volumes of CO₂ (to achieve 0, 0.5, 1.0, 2.0 % v/v).
• Constant light source (e.g., 1500 lux, white).
• Thermometer (maintained at 25 °C).
• Stopwatch.

7.2 Method

1. Set light intensity to a fixed value (e.g., 1500 lux) and allow the system to stabilise for 2 min.
2. Introduce CO₂ to obtain the required concentration; mix gently and allow 2 min for equilibration.
3. Start gas collection and record O₂ volume every minute for 10 min.
4. Repeat for each CO₂ level, keeping temperature and light constant.
5. Carry out three replicates per concentration; randomise the order of treatments.

7.3 Sample Data Table

7.4 Analysis

• Calculate the rate of O₂ evolution for each CO₂ concentration (mL min⁻¹).
• Graph rate versus CO₂ concentration. The curve is typically hyperbolic and can be described by the Michaelis–Menten equation:

  
  

  \$\text{Rate} = \frac{V{\max}\,[\text{CO}2]}{Km + [\text{CO}2]}\$

  • Kₘ = CO₂ concentration at half‑maximal rate (reflects Rubisco affinity).
  • Vₘₐₓ = maximum rate when Rubisco is saturated.

• Discuss differences between C₃ and C₄ plants:

  • C₃ plants (e.g., spinach) show a pronounced CO₂‑limited region because Rubisco also catalyses photorespiration.
  • C₄ plants (e.g., maize) possess a CO₂‑concentrating mechanism; the curve reaches Vₘₐₓ at lower external CO₂ and is less sensitive to ambient CO₂ changes.

8. Investigating Temperature

8.1 Apparatus

• Whole Elodea sprigs in a thermostatically controlled water bath.
• Bath temperatures: 10 °C, 20 °C, 30 °C, 40 °C (±0.5 °C).
• Constant light source (1500 lux, white).
• Inverted graduated cylinder for O₂ collection.
• Thermometer and timer.

8.2 Method

1. Place the plant in the water bath; allow 5 min for the tissue to reach the target temperature.
2. Start gas collection and record O₂ volume each minute for 10 min.
3. Repeat at each temperature while keeping light intensity and CO₂ concentration constant (ambient air ≈0.04 % v/v CO₂).
4. Perform three replicates per temperature; randomise the sequence of temperatures.

8.3 Sample Data Table

8.4 Analysis

• Determine the rate of O₂ evolution for each temperature.
• Plot rate versus temperature. Expected pattern:

  • Rate rises with temperature (Q₁₀ effect – roughly doubles for each 10 °C increase) up to an optimum (~30 °C for most temperate C₃ plants).
  • Beyond the optimum the rate declines because enzymes (especially Rubisco) denature and membrane fluidity is compromised.

• Explain the decline in terms of enzyme kinetics (loss of active sites) and membrane stability.

9. Integrating the Three Limiting Factors

After completing the three single‑factor investigations, compare the slopes of the following graphs (all measured under the same baseline conditions: 25 °C, ambient CO₂, 1500 lux):

• Rate vs Light intensity
• Rate vs CO₂ concentration
• Rate vs Temperature

The factor that shows the smallest slope or reaches a plateau first is the limiting factor for that particular set‑up. In natural environments the limiting factor can shift (e.g., light‑limited under a dense canopy, temperature‑limited at high altitude).

10. Data Analysis – Identifying the Limiting Factor (Rubric)

1. Calculate the mean rate (±SD) for each treatment.
2. Construct three separate graphs (rate vs each factor).
3. Determine:

   • Slope of the linear region (steepness indicates sensitivity).
   • Point of plateau (where increasing the factor no longer raises the rate).

4. Compare the slopes/plateau points:

   • If the light curve plateaus while CO₂ and temperature curves are still rising, light is limiting.
   • If the CO₂ curve plateaus first, CO₂ is limiting.
   • If the temperature curve shows a peak and then declines while the other two are still increasing, temperature is limiting.

5. State the limiting factor clearly and justify with reference to the graphs and calculated values.

11. Statistical Considerations

• Minimum of three replicates per treatment; calculate mean, standard deviation and, where appropriate, standard error.
• Randomise the order of treatments to avoid systematic drift (e.g., gradual warming of the water bath).
• If the class has access to statistical software, a one‑way ANOVA can be used to test for significant differences between treatment means (p < 0.05).

12. Evaluation of Reliability

• Sources of random error: Bubbles escaping, temperature fluctuations, inconsistent leaf area.
• Sources of systematic error: Mis‑calibrated lux meter, CO₂ leakage, light heat affecting temperature.
• Suggested improvements:

  • Use a digital light sensor with data‑logging capability.
  • Employ a gas‑tight chamber with a septum for CO₂ injection to prevent leaks.
  • Measure leaf area with a grid or image‑analysis software and keep it constant.
  • Include a blank control (no plant) to account for any background gas changes.

13. Safety Considerations

• Wear insulated gloves when handling hot water baths.
• Secure CO₂ cylinders; use regulators rated for the pressure of the apparatus and work in a well‑ventilated area.
• Protect eyes from bright lamps (especially when using UV‑rich sources).
• Handle glassware carefully to avoid breakage and possible injury.

14. Real‑World Application Box